Project Summary Transcriptome turnover is the process by which an organ system or cell type gains and loses the expression of genes over evolutionary time. Comparative data indicate that gains and losses of genes within a tissue-specific transcriptome are widespread phenomena that can have a large impact on tissue function and physiological characteristics of organisms. Genes enter and leave the tissue transcriptome through two classes of genomic changes: structural and regulatory. Structural changes are changes in genome content that occur through processes such as gene duplication and deletion. Regulatory changes are changes in the tissue- specific expression of conserved genes, which occur through the gain or loss of non-coding sequences that control the expression of genes. These regulatory alterations happen through either cis (mutations in non- coding sequences that regulate a gene directly) or trans changes (mutations that alter an upstream regulating protein such as a transcription factor). The mechanism by which genes turnover in the tissue transcriptome may predict their likelihood of cooption into new regulatory circuits in the new tissue and of playing an adaptive role in the new transcriptome. This research seeks to investigate the relative rates of structural and regulatory turnover and the relative essentiality of genes gained through these mutational processes. Comparative transcriptome profiles, evolutionary models and statistical analysis will be used to estimate the relative frequency of structural versus regulatory turnover in rapidly evolving reproductive organs in 11 species in the D. melanogater species group: the structurally complex testes and structurally simple accessory glands. To compare their essentiality expression of genes that gained expression through regulatory versus structural mechanisms will be knocked down in D. melanogaster accessory glands to directly compare their essentiality to organ function. The impact of transcription factor cooption on the transcriptome and phenotype of the accessory glands will be investigated through transgenic techniques. The outcome of this research will be an understanding of the frequency and phenotypic impact of genomic mutation types that contribute to transcriptome turnover. This will provide a more detailed understanding of which mutational processes may be the most important sources of new adaptive genetic materials for evolving organs. Characterizing the rate and phenotypic impact of the forces moving genes in and out of organ transcriptomes?whether structural or regulatory; cis or trans?will also allow researchers to use comparative transcriptomics data to home in on the functionally important aspects of the transcriptome when studying human health and disease.